Stabilized formulations of alpha adrenergic receptor antagonists and the uses thereof

ABSTRACT

The present invention provides compositions and stable liquid formulations comprising alpha adrenergic receptor antagonists and use thereof for increasing blood flow. In one embodiment, the stable liquid formulations of this invention are useful for reversing the effects of an anesthetic agent, preferably a long-lasting local anesthetic agent administered in conjunction with an alpha adrenergic receptor agonist.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is in the field of pharmaceutical chemistry. The inventionrelates in particular to a method of reversing local anesthesia inducedby a local anesthetic and an alpha-adrenergic agonist, comprisingadministering an effective low dose of a stabilized liquid formulationof an alpha-adrenergic antagonist.

2. Related Art

Local anesthesia is widely used by dentists to provide pain relief topatients during dental procedures. To provide pain relief, a drugformulation containing a local anesthetic compound such as lidocaine isinjected into the gum tissue surrounding the tooth or teeth on which thedental procedure is to be performed. There are short-acting andlong-lasting local anesthetic drug formulations. Short-acting localanesthetic drug formulations contain lidocaine or a related localanesthetic drug dissolved in saline or other suitable injection vehicle.Typically, local anesthesia with short-acting local anesthetics lastsapproximately 20-30 minutes, which is not long enough for many dentalprocedures. To obtain long-lasting local anesthesia, dentists often uselidocaine or other local anesthetic formulations which, in addition tothe local anesthetic drug itself, contain low concentrations ofepinephrine or another adrenergic receptor agonist such aslevonordefrin. More than 90% of the local anesthesia proceduresperformed by dentists involve local anesthetic formulations containingalpha adrenergic receptor agonists. The vasoconstrictor is necessarybecause local anesthetics without vasoconstrictor are too short-actingfor most dental procedures. The added epinephrine stimulates alphaadrenergic receptors on the blood vessels in the injected tissue. Thishas the effect of constricting the blood vessels in the tissue. Theblood vessel constriction causes the local anesthetic to stay in thetissue much longer, resulting in a large increase in the duration of theanesthetic effect (from 20 minutes for the short-acting formulation to3-6 hours for the long-lasting formulation). A major problem with theuse of epinephrine-containing local anesthetics is soft-tissueanesthesia (lip, cheek, tongue) which usually lasts many hours longerthan anesthesia and analgesia of the tooth pulp. Tooth pulp anesthesiaand analgesia are the desired effects of local anesthesia from a dentalprocedural perspective while soft-tissue anesthesia is usually anundesirable side effect. Soft tissue anesthesia results in a number ofproblems and inconveniences, such as a prolonged and uncomfortablefeeling of numbness in and around the mouth, inability to smile,difficulty eating, drinking and swallowing, loss of productivity bymissing work hours or meetings etc. Lingering soft-tissue anesthesia canbe the cause of injuries due to biting of the tongue or lips.Furthermore, lingering soft-tissue anesthesia is an inconvenience and itis perceived as an annoyance by many patients. Lingering soft-tissueanesthesia can lead to injury especially in children who often bite intothe anesthetized tissue out of curiosity. It would therefore bedesirable to have a drug that could be used at will by dentists torapidly reverse local anesthesia after it is no longer needed

U.S. Pat. No. 4,659,714 discloses a method of prolonging localanesthesia by coadministering a vasoconstrictor, in particular, avasoconstrictor that acts upon the alpha adrenergic receptor sites ofthe blood vessel walls. The '714 patent also discloses the subsequentadministration of an alpha adrenergic receptor antagonist to causereduction of the prolonged anesthesia effect. Included within the groupof alpha adrenergic receptor antagonists described in this patent arephentolamine mesylate. However, the examples make reference to theadministration of “phentolamine.” It is much more likely that what wasadministered was phentolamine mesylate since phentolamine mesylate isFDA approved and readily soluble in water. In contrast, phentolamine isnot FDA approved and is relatively insoluble in water.

As shown in Example 1, Table 1 of the '714 patent, 0.5-1.5 mg of“phentolamine” was administered to groups of patients which werepretreated with lignocaine admixed with epinephrine. The results inTable 1 show a reduction in the duration of anesthesia with increasingamounts of “phentolamine.” In Example 2, 2 mg of “phentolamine” wasadministered. In Example 3, four injections of 1 mg each (4 mg total) of“phentolamine” were administered. In Example 4, four injections of 1 mgeach (4 mg total) of “phentolamine” were administered.

The drug doses of “phentolamine” described in the '714 patent (0.5-4 mg)overlap the doses of phentolamine mesylate that are approved by the FDAfor the systemic treatment of high blood pressure in patients withpheochromocytoma (total dose of 5 mg in a solution of 2.5-5 mg/ml).Since those doses are normally intended for systemic treatment of highblood pressure, those high dose levels can cause severe side effectswhen used in healthy, normal people. The package insert of thephentolamine mesylate product states the following side effect warning:“Myocardial infarction, cerebrovascular spasm, and cerebrovascularocclusion have been reported to occur following the administration ofphentolamine, usually in association with marked hypotensive episodes.”Thus, the drug doses taught by the '714 patent for the reversal of localanesthesia may cause unacceptable side effects, precluding the use ofthis product for anesthesia reversal in healthy normal subjects in adentist's office.

It has been discovered that a highly effective local anesthesia reversalcan be obtained by injections of much lower concentrations ofphentolamine mesylate than is disclosed in the '714 patent. See WO01/85171. It has been found that a solution containing only 0.05 mg/mlof phentolamine mesylate can rapidly reverse the effect of a localanesthetic containing an alpha adrenergic receptor agonist. Thisphentolamine mesylate drug concentration is 20-100 times lower than thephentolamine mesylate drug concentration taught by the '714 patent. Theadvantage is that, at such low phentolamine mesylate drugconcentrations, no systemic side effects such as myocardial infarctionand cerebrovascular spasm will be observed. This allows the safe andeffective use of phentolamine mesylate for local anesthesia reversalwithout causing life-threatening or other untoward side effects. Indeed,in a human clinical efficacy study using a low concentration formulationof phentolamine mesylate, a highly effective anesthesia reversal wasobserved without any side-effects whatsoever. This constitutes a crucialimprovement of the local anesthesia reversal method taught by the '714patent.

The present invention is directed to the discovery that prior artformulations of phentolamine mesylate are unstable in water and can notbe stored reconstituted in water or saline. Stable liquid formulationshave also been discovered which allow prolonged storage of phentolaminemesylate.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and stable liquidformulations comprising alpha adrenergic receptor antagonists and usethereof for increasing blood flow. In one embodiment, the stable liquidformulations of this invention are useful for reversing the effects ofan anesthetic agent, preferably a long-lasting local anesthetic agentadministered in conjunction with an alpha adrenergic receptor agonist.

In one embodiment, the invention relates to a method of increasing bloodflow in a mammal, comprising administering to mammal a stable liquidformulation comprising an alpha adrenergic receptor antagonist.

In a preferred embodiment, the administration of a stable liquidformulation comprising an alpha adrenergic receptor antagonist is usedto counteract a prior administration of an alpha adrenergic receptoragonist.

In another embodiment, the invention relates to a method of providinglocal anesthesia to a mammal, comprising:

-   -   (a) administering to the mammal in need thereof an anesthetic        agent in an amount effective to provide local anesthesia, and        then    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        time of anesthesia.

In a preferred embodiment, the invention relates to a method ofproviding local anesthesia to a mammal, comprising:

-   -   (a) administering to the mammal in need thereof an anesthetic        agent and an alpha adrenergic receptor agonist to the site to be        anesthetized, wherein said anesthetic agent is administered in        an amount effective to provide local anesthesia and said alpha        adrenergic receptor agonist is administered in an amount        effective to constrict the blood vessels at the site and prolong        the local anesthesia, and then    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation.

In a more preferred embodiment, the invention relates to a method ofproviding local anesthesia to a human, comprising:

-   -   (a) administering to a human in need thereof by injection to the        site to be anesthetized a solution comprising polocaine and        levonordefrin, wherein said polocaine is administered in an        amount effective to provide local anesthesia and said        levonordefrin is administered in an amount effective to        constrict the blood vessels at the site and prolong the local        anesthesia, thereby producing local anesthesia at said site,    -   (b) carrying out a medical procedure on the human, and then    -   (c) administering a stable liquid formulation comprising        phentolamine mesylate at said site at a concentration of about        0.1 mg/ml or less to reduce the prolongation.

The invention also relates to a method of enhancing the survival of atissue graft, comprising

-   -   (a) administering to a mammal undergoing a tissue graft an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the tissue graft, wherein said anesthetic agent is        administered in an amount effective to provide local anesthesia        and said alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the tissue graft procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and enhance the tissue graft survival.

The invention also relates to a method of reducing the occurrence ofdermal necrosis during a medical procedure, comprising

-   -   (a) administering to a mammal undergoing a medical procedure an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the procedure, wherein said anesthetic agent is        administered in an amount effective to provide local anesthesia        and said alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the medical procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and reduce the occurrence of dermal necrosis during        the procedure.

This invention also relates to a method of treating a trigger point in amammal, comprising:

-   -   (a) performing a trigger point injection in a mammal having a        trigger point, optionally with administration of an anesthetic        agent, and    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to increase        blood flow to the area of the trigger point and enhance the        treatment of the trigger point.

The invention also relates to a method of providing a regionalanesthetic block to a mammal, comprising:

-   -   (a) administering to the mammal in need thereof an anesthetic        agent and an alpha adrenergic receptor agonist in the site to        receive the anesthetic block, wherein said anesthetic agent is        administered in an amount effective to provide local anesthesia        and said alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels in the site and        prolong the anesthetic block, and then    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation.

This invention also relates to a method of decreasing the occurrence ofdry socket, comprising:

-   -   (a) administering to a mammal undergoing a tooth extraction an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the tooth extraction, wherein the anesthetic agent is        administered in an amount effective to provide local anesthesia        and the alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the tooth extraction procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and decrease the occurrence of dry socket.

This invention also relates to a method of enhancing the survival of aninjured or diseased tooth in a method, comprising:

-   -   (a) administering to a mammal undergoing repair of an injured        tooth or treatment of a diseased tooth an anesthetic agent and        an alpha adrenergic receptor agonist to the site of the tooth,        wherein the anesthetic agent is administered in an amount        effective to provide local anesthesia and the alpha adrenergic        receptor agonist is administered in an amount effective to        constrict the blood vessels at the site and prolong the local        anesthesia,    -   (b) performing the repair or treatment procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and enhance the survival of the injured or diseased        tooth.

This invention also relates to a method for the treatment of periodontaldisease, comprising administering to a mammal having periodontal diseasea stable liquid formulation comprising an alpha adrenergic receptorantagonist to said site to increase gingival blood flow and enhance thetreatment of the periodontal disease.

The invention also relates to a stable liquid formulation comprising analpha adrenergic receptor antagonist. The formulation may containadditives such as metal chelators and tonicity modifiers which enhancethe stability of the alpha adrenergic receptor antagonist and allowstorage of the antagonist for long periods (e.g., greater than 12months).

The invention also relates to a stable liquid formulation comprisingphentolamine mesylate.

The invention also relates to a kit comprising a carrier means having inclose confinement therein two or more container means, wherein a firstcontainer means comprises an anesthetic agent and optionally an alphaadrenergic receptor agonist and a second container means comprises astable liquid formulation comprising an alpha adrenergic receptorantagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a wide range pH stability profile of phentolamine mesylateat 40° C.

FIG. 2 shows a narrow range pH stability profile of phentolaminemesylate at 60° C.

FIG. 3 shows the effect of administration of phentolamine mesylate onthe time to return to normal sensation in anesthetized tissues afterinferior alveolar nerve block.

FIG. 4 shows a dose response study of the effect of administration ofphentolamine mesylate on the time to return to normal sensation inanesthetized tissues after inferior alveolar nerve block.

FIG. 5 shows a dose response study of the effect of administration ofphentolamine mesylate on the time to return to normal sensation inanesthetized tissues after lateral incisor infiltration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and stable liquidformulations comprising alpha adrenergic receptor antagonists and usethereof for increasing blood flow. In one embodiment, the inventionrelates to a method of increasing blood flow in a mammal, comprisingadministering to the mammal a stable liquid formulation comprising analpha adrenergic receptor antagonist. In one embodiment of theinvention, the increased blood flow is within a specific tissue orportion of the body of the mammal to which the stable liquid formulationhas been administered. In another embodiment, the increased blood flowis systemic.

Preferably, the administration of a stable liquid formulation comprisingan alpha adrenergic receptor antagonist is used to counteract a prioradministration of an alpha adrenergic receptor agonist.

In another embodiment, the invention relates to a method of providinglocal anesthesia to a mammal, comprising:

-   -   (a) administering to the mammal in need thereof an anesthetic        agent in an amount effective to provide local anesthesia, and        then    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        time of anesthesia.

In a preferred embodiment, the invention relates to a method ofproviding local anesthesia to a mammal, comprising:

-   -   (a) administering to the mammal in need thereof an anesthetic        agent and an alpha adrenergic receptor agonist to the site to be        anesthetized, wherein said anesthetic agent is administered in        an amount effective to provide local anesthesia and said an        alpha adrenergic receptor agonist is administered in an amount        effective to constrict the blood vessels at the site and prolong        the local anesthesia, and then    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation.

The anesthetic agent and alpha adrenergic receptor agonist may beadministered together as part of a unitary pharmaceutical composition oras part of separate pharmaceutical compositions so long as the alphaadrenergic receptor agonist acts to constrict the blood vessels in thevicinity of where the anesthetic agent has been administered to resultin a prolonging of anesthesia. In a preferred embodiment, the anestheticagent and alpha adrenergic receptor agonist are administered together insolution. The anesthetic agent and alpha adrenergic agonist may beadministered by injection, by infiltration or by topical administration,e.g. as part of a gel or paste.

In a preferred embodiment, a solution comprising the anesthetic agentand alpha adrenergic receptor agonist is administered by injectiondirectly into the site to be anesthetized, e.g. prior to a dentalprocedure.

Examples of local anesthetics that may be used in the practice of theinvention include without limitation lidocaine, polocaine, lignocaine,xylocaine, novocaine, carbocaine, etidocaine, procaine, prilocaine,bupivacaine, cinchocaine and mepivacaine.

Examples of alpha adrenergic receptor agonists that can be usedaccording to the invention include catecholamines and catecholaminederivatives. Particular examples include without limitationlevonordefrin, epinephrine, and norepinephrine.

Examples of alpha adrenergic receptor antagonists that can be used inthe practice of the invention include without limitation phentolamine,phentolamine hydrochloride, phentolamine mesylate, tolazoline,yohimbine, rauwolscine, doxazosine, labetalol, prazosine, tetrazosineand trimazosine. Phentolamine mesylate is approved by the FDA for thetreatment of hypertension in patients with pheochromocytoma, for thetreatment of dermal necrosis and sloughing following accidentalextravasation of norepinephrine, and for the diagnosis ofpheochromocytoma (phentolamine blocking test). Phentolamine mesylate issupplied as a lyophilized formulation comprising mannitol in vialscontaining 5 mg of drug substance which may be dissolved inphysiological saline or other pharmaceutically acceptable carrier.

In order to reverse the local anesthesia after a medical procedureaccording to the present invention, the alpha adrenergic receptorantagonist is preferably administered at a low dose, i.e. at a dose thatdoes not cause side effects, i.e. at or below about 0.45 mg per dose foradults (at or below about 0.0064 mg/kg) or 0.18 mg per dose forchildren, more preferably below about 0.25 mg per dose for adults (at orbelow about 0.0036 mg/kg) or 0.1 mg per dose for children, morepreferably, below about 0.1 mg per dose for adults (below about 0.0014mg/kg) or 0.04 mg per dose for children, most preferably, at about 0.08mg per dose for adults (about 0.0011 mg/kg) or about 0.032 mg per dosefor children, of phentolamine mesylate or a molar equivalent of anotheradrenergic receptor antagonist. In a preferred embodiment, the alphaadrenergic receptor antagonist is present at a concentration of about 1mg/ml or less, preferably from about 0.001 mg/ml to about 0.25 mg/ml,more preferably, about 0.05 mg/ml to about 0.1 mg/ml.

The alpha adrenergic receptor antagonist may be administered byinjection into the site of anesthesia, by infiltration or by topicaladministration. In a preferred embodiment, the alpha adrenergic receptorantagonist is administered to mucosal tissue. In this embodiment, thealpha adrenergic receptor antagonist may be applied to the site in theform of an impregnated wafer, pellet or cotton ball, whereby theantagonist is taken up by the mucosal tissue resulting in reversal ofthe anesthesia. In another embodiment, the alpha adrenergic receptorantagonist is administered to the site of a regional anesthetic block toreverse the block, e.g. by injection or infiltration into the site. In apreferred embodiment, the alpha adrenergic receptor antagonist isadministered via a cannula into the epidural space of an animal toreverse epidural anesthesia.

Examples of medical procedures that may be carried out according to thepresent invention include, without limitation, both major and minorsurgery, dental procedures, cosmetic surgery, tissue grafting (e.g. hairand bone grafting) and cesarean section. In one embodiment, reversal ofanesthesia according to the present invention is carried out by medicaltrainees to mitigate any mistakes that are made, and which may lead tothe loss of extremities such as fingers, as well as ears and tips ofnoses.

Hyaluronidase, an enzyme which enhances the diffusion of drugs withintissues, may be administered together with the alpha adrenergic receptorantagonist. The hyaluronidase and alpha adrenergic receptor antagonistmay be administered together as part of a unitary pharmaceuticalcomposition or as part of separate pharmaceutical compositions, so longas the hyaluronidase and alpha adrenergic receptor antagonist areadministered to the site where anesthesia is to be reversed and arepresent in amounts effective to enhance the diffusion of the alphaadrenergic receptor antagonist and to reverse the anesthesia,respectively. The hyaluronidase is administered one or more times intothe site of anesthesia. In general, about 1.5 U to about 200 U ofhyaluronidase is administered in one or more injections. In a mostpreferred embodiment, about 200 U of hyaluronidase is administered byinjection into the site. Those of ordinary skill in the art candetermine optimal amounts of hyaluronidase with no more than routineexperimentation.

When performing hair grafts, the surgeon often injects an anesthetic andepinephrine to reduce bleeding and provide a clear vision of the site.According to Bernstein, R. M. and Rassman, W. R., Hair Transplant ForumInternational 10:39-42 (2000), the usefulness of epinephrine in hairgraft procedures is limited by a number of factors includingpost-operative telogen effluvium when epinephrine is used in largetransplant sessions. In addition, when adrenaline is added to an areawhose blood supply is already compromised by a large number of recipientsites, the tissue may not receive enough oxygen. Although not proven,according to Bernstein and Rassman it is likely that epinephrineinfiltration into the recipient area is a contributing factor in thedevelopment of the “central necrosis” that has occasionally beenreported during hair transplantation. Furthermore, it is possible thatthe intense vasoconstrictive action of epinephrine may contribute to thedecreased graft survival. Thus, according to the present invention, onemay achieve enhanced tissue graft survival in a method comprising:

-   -   (a) administering to a mammal undergoing a tissue graft an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the tissue graft, wherein the anesthetic agent is        administered in an amount effective to provide local anesthesia        and the alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the tissue graft procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and enhance the tissue graft survival.

In a preferred embodiment, the tissue graft is a hair graft. In anotherpreferred embodiment, a low dose of alpha adrenergic receptor antagonistis administered to the site to avoid untoward side effects.

Such hair grafts include skin flaps containing a plurality of hair cellsand single transplanted hair cell follicles. Typically, such hair graftsare obtained from a site on the animal that has actively growing hair.According to the present invention, an alpha adrenergic receptorantagonist is administered after a hair graft procedure to reverse thelocal anesthesia and reduce post-operative telogen effluvium (sheddingof hair) and survival of the skin flap.

In another embodiment, hyaluronidase may be administered to the tissuegraft site to increase survival of the graft. According to Pimentel, L.A. S. and Goldenburg, R. C. d. S, Revista da Soociedade Brasileira deCirurgia Plastica 14 (1999), the local administration of hyaluronidaseincreases skin flap survival. According to the authors, hyaluronidase isan enzyme that reduces or prevents tissue injury presumably by causingthe rapid diffusion of extravasated fluids to distant areas, thusallowing a better turnover of nutrients. The hyaluronidase is generallyinjected one or more times into the site of the hair graft. Similarly,the present invention can be used to improve survival of other engraftedtissues or bone in any graft surgical procedure where a local anestheticand an alpha adrenergic receptor agonist is used to minimize bleedingduring the surgery and where subsequent rapid reperfusion of tissue isdesired in order to increase graft survival.

In a further preferred embodiment, the tissue graft is a dental implant.According to the present invention, an alpha adrenergic receptorantagonist is administered after a dental implant procedure to reversethe local anesthesia, increase blood flow to the involved area andpromote the survival of the implant.

When local anesthesia is used for medical procedures one potentialside-effect is dermal necrosis due to the decreased blood flow to theanesthetized area. Rapid reversal of the anesthesia after the procedureis finished would result in increased blood flow and therefore anincreased supply of oxygen to the affected tissue. Thus, in anadditional embodiment, one may decrease the occurrence of dermalnecrosis during a medical procedure in a method comprising:

-   -   (a) administering to a mammal undergoing a medical procedure an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the procedure, wherein said anesthetic agent is        administered in an amount effective to provide local anesthesia        and said alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the medical procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and reduce the occurrence of dermal necrosis during        the procedure.

In a preferred embodiment, a low dose of the alpha adrenergic receptorantagonist is administered.

An increasing number of people are carrying and using autoinjectors foremergencies in which rapid treatment of symptoms is necessary. Forexample, people with severe allergies to bee stings and the likefrequently carry an autoinjector containing epinephrine for immediateuse when they are stung. The increased use of autoinjectors has led toan increase in the number of accidental needle sticks with theautoinjectors, particularly in the fingertip. Such accidental injectionof epinephrine leads to a significant decrease in blood flow to thefinger, resulting in tissue necrosis, and, potentially, loss of thefinger. Thus, in a further embodiment of the invention, needle stickswith a vasoconstrictor, such as epinephrine, are treated byadministering a stable liquid formulation comprising an alpha adrenergicreceptor antagonist to the site of the needle stick to reduce theoccurrence of tissue necrosis.

Trigger points are discrete, focal, hyperirritable spots located in ataut band of skeletal muscle. Alverez, D. J. and Rockwell, P. G., Amer.Fam. Physician 65:653-660, 2002. Trigger points produce local andreferred pain. Needling of the trigger point, either dry or concomitantwith injection of a local anesthetic is one of the most effectivetreatments to inactivate the trigger point and relieve the symptoms.Reperfusion of the trigger point is also thought to provide pain relief.It is therefore advantageous to enhance blood flow to the trigger pointduring or after injection. Thus, according to the present invention, onemay enhance the beneficial effect of trigger point injection in a methodcomprising:

-   -   (a) performing a trigger point injection in a mammal having a        trigger point, optionally with administration of an anesthetic        agent, and    -   (b) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist at the site of the trigger        point to increase blood flow to the area of the trigger point        and enhance the treatment of the trigger point.

When an anesthetic agent is injected into the trigger point, an alphaadrenergic receptor agonist may also be injected in an amount effectiveto constrict the blood vessels at the site and prolong the localanesthesia. In a preferred embodiment, a low dose of the alphaadrenergic receptor antagonist is administered.

In a further embodiment, a stable liquid formulation of an alphaadrenergic receptor antagonist is administered after a regionalanesthetic block to reverse the block. Epidural anesthesia is commonlyadministered to provide a regional anesthetic block in a number ofmedical procedures including child birth, cesarean section, surgery tothe pelvis and the like. Prolonged epidural anesthesia has many untowardside effects, including prolonged paralysis, inability to voluntarilyurinate, and hypotension. Typically, the anesthesiologist injects intothe epidural space an equal volume of saline in an effort to dilute theanesthetic and reduce the anesthesia.

The present invention solves the side-effect problems by providing foron demand reversal of the anesthesia without the need for injectinglarge volumes of saline. In this embodiment, the invention relates to amethod of providing a regional anesthetic block to a mammal, comprising:

-   -   (a) administering to a mammal in need thereof an anesthetic        agent and an alpha adrenergic receptor agonist in the site to        receive the anesthetic block, wherein the anesthetic agent is        administered in an amount effective to provide local anesthesia        and the alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels in the site and        prolong the local anesthesia, and then    -   (b) administering a stable liquid formulation of an alpha        adrenergic receptor antagonist to the site to reduce the        prolongation.

In a preferred embodiment, a low dose of the alpha adrenergic receptorantagonist is administered. In another preferred embodiment, theanesthetic block is epidural anesthesia and the site of the block is theepidural space. The invention has application to reversal of otherblocks as well including brachial plexus and femoral blocks.

In another embodiment, hyaluronidase is administered together with thealpha adrenergic receptor antagonist to enhance the diffusion of thealpha adrenergic receptor antagonist within the site of the block, e.g.the epidural space, and speed reversal of the anesthesia.

When a local anesthetic comprising an alpha adrenergic receptor agonistis administered for a dental procedure there is a significant decreasein both gingival and pulpal blood flow which can last for extendedperiods of time (greater than one hour). Pogrol, A. J., Oral Surg. OralMed. Oral Pathol. Oral Radiol. Endod. 85:197-202, 1998. Thus, in manydental situations in which blood flow is critical to the continuedhealth of the tooth and/or surrounding tissue, it is advantageous toreverse the effect of the alpha adrenergic receptor agonist as soon aspossible after the anesthetic is no longer needed.

When a tooth is extracted, the empty socket fills with blood and a clotis formed. If the clot is prematurely lost or degraded, an extremelypainful condition develops due to the exposure of the bone and nerveendings. This condition is known as alveolar osteitis or dry socket. Theoccurrence of dry socket has been shown to increase when the socket isonly partially filled with blood following extraction. Heasman, P. A.and Jacobs, D. J., Br. J. Oral Maxillofacial Surg. 22:115-122, 1984. Byreversing local anesthesia after the extraction procedure is completed,blood flow to the area around the extraction socket will increase,leading to enhanced filling of the socket. Thus, according to thepresent invention, one may decrease the occurrence of dry socket in amethod comprising:

-   -   (a) administering to a mammal undergoing a tooth extraction an        anesthetic agent and an alpha adrenergic receptor agonist to the        site of the tooth extraction, wherein the anesthetic agent is        administered in an amount effective to provide local anesthesia        and the alpha adrenergic receptor agonist is administered in an        amount effective to constrict the blood vessels at the site and        prolong the local anesthesia,    -   (b) performing the tooth extraction procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and decrease the occurrence of dry socket.

In a preferred embodiment, a low dose of the alpha adrenergic receptorantagonist is administered.

When traumatic injury to a tooth or tooth disease occurs, necrosis ofthe pulp tissue can result, often leading to tooth loss. To sustain thevitality of the pulp, blood flow must be maintained andrevascularization must occur. By reversing local anesthesia after repairof the injured tooth or treatment of the tooth disease is completed,blood flow to the area of the tooth will increase, leading to enhancedblood flow to the necrotic tissue. Thus, according to the presentinvention, one may enhance the survival of an injured or diseased toothin a method comprising:

-   -   (a) administering to a mammal undergoing repair of an injured        tooth or treatment of a diseased tooth an anesthetic agent and        an alpha adrenergic receptor agonist to the site of the tooth,        wherein the anesthetic agent is administered in an amount        effective to provide local anesthesia and the alpha adrenergic        receptor agonist is administered in an amount effective to        constrict the blood vessels at the site and prolong the local        anesthesia,    -   (b) performing the repair or treatment procedure, and then    -   (c) administering a stable liquid formulation comprising an        alpha adrenergic receptor antagonist to said site to reduce the        prolongation and enhance the survival of the injured or diseased        tooth.

In a preferred embodiment, a low dose of the alpha adrenergic receptorantagonist is administered.

As discussed above, when a local anesthetic comprising an alphaadrenergic receptor agonist is administered for a dental procedure thereis a significant decrease in both gingival and pulpal blood flow whichcan last for extended periods of time (greater than one hour). Underthese circumstances, jaw muscles become sore for being open for so long.This soreness is due to the jaw muscles being tired and cramped. Musclespasm is accentuated by the use of the agonist. The use of an alphaadrenergic antagonist causes rapid localized reperfusion of blood thusreducing or eliminating muscle spasm and post operative pain. In thisembodiment, the invention relates to a method of reducing or eliminatingmuscle spasm or post operative pain, comprising:

-   -   (a) administering to a mammal in need thereof an anesthetic        agent and an alpha adrenergic receptor agonist in a site to be        anesthetized, wherein the anesthetic agent is administered in an        amount effective to provide local anesthesia and the alpha        adrenergic receptor agonist is administered in an amount        effective to constrict the blood vessels in the site and prolong        the local anesthesia, and then    -   (b) administering a stable liquid formulation of an alpha        adrenergic receptor antagonist to the site to reduce or        eliminate muscle spasm or post operative pain at the site.

When the method is to reduce or eliminate post operative pain, a medicalprocedure (such as a dental procedure) may be performed after (a) andbefore (b).

The invention also provides for a method of reducing muscle spasm ingeneral such as muscle spasm associated with headache. In thisembodiment, an alpha adrenergic receptor antagonist is administered tothe site of muscle spasm in an amount effective to reduce or eliminatethe muscle spasm. This aspect of the present invention has particularapplication to the treatment of tension headaches caused by musclespasm. The alpha adrenergic receptor antagonist may be administered inany way that achieves the intended purpose, e.g. by injection or topicalapplication to the site of muscle spasm.

Periodontal disease is a disease of the gums in which the gums areinflamed and/or bleeding due to the presence of bacteria. Poor gingivalblood flow exacerbates periodontal problems. Tobacco smokers haveincreased periodontal disease, in part due to the fact that smokingdecreases gingival blood flow. One of the consequences of periodontaldisease is the formation of pockets between the gum and the tooth wherethe attachment of the gum to the tooth is lost. Treatment of periodontaldisease often involves irrigation of periodontal pockets with waterand/or medicines to clean and treat the gums. It is advantageous toincrease gingival blood flow for the treatment of periodontal disease.Thus, according to the present invention, one may treat periodontaldisease in a method comprising administering to a mammal havingperiodontal disease a stable liquid formulation comprising an alphaadrenergic receptor antagonist at the site of the periodontal disease toincrease gingival blood flow, thereby treating the periodontal disease.The alpha adrenergic receptor antagonist can be administered as part ofan irrigant for periodontal pockets. In a preferred embodiment, a lowdose of the alpha adrenergic receptor antagonist is administered. Thealpha adrenergic receptor antagonist can be administered afteradministration of an anesthetic agent and an alpha adrenergic receptoragonist to the site of the tooth, wherein the anesthetic agent isadministered in an amount effective to provide local anesthesia and thealpha adrenergic receptor agonist is administered in an amount effectiveto constrict the blood vessels at the site and prolong the localanesthesia. The local anesthetic can be administered for a dentalprocedure related to the periodontal disease, e.g., deep scaling or gumsurgery.

The invention also relates to stable liquid formulations comprisingalpha adrenergic receptor antagonists, particularly low doseformulations. A “stable liquid formulation” is defined as one in whichthe alpha adrenergic receptor antagonist is solubilized and wherein theconcentration and purity of the alpha adrenergic receptor antagonist is90% or greater, preferably 95% or greater, after storage at 2-40° C.,preferably refrigeration temperature (2-8° C.) or room temperature (25°C.) for at least 3 months, preferably at least 6 months, more preferablyat least 12 months.

Examples of alpha adrenergic receptor antagonists that can be used inthe stable liquid formulations of this invention include withoutlimitation phentolamine, tolazoline, yohimbine, rauwolscine, doxazosine,labetalol, prazosine, tetrazosine and trimazosine, as well aspharmaceutically acceptable salts of any of the above. In a preferredembodiment, the alpha adrenergic receptor antagonist is a salt ofphentolamine. More preferably, the alpha adrenergic receptor antagonistis selected from phentolamine hydrochloride or phentolamine mesylate.Most preferably, the alpha adrenergic receptor antagonist isphentolamine mesylate.

Pharmaceutically acceptable salts of the alpha adrenergic receptorantagonist utilized in the stable liquid formulations of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate,oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate,picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The stable liquid formulation of the present invention may comprise thealpha adrenergic receptor antagonist at any concentration up to thelimit of its solubility. This is typically in the range of 0.01 mg/ml toat least 10 mg/ml. In a preferred formulation, the alpha adrenergicreceptor antagonist is phentolamine mesylate which is preferably presentat a concentration of about 0.01 mg/ml to about 1 mg/ml in the stableliquid formulation, more preferably about 0.01 mg/ml to about 0.25mg/ml, and most preferably about 0.1 mg/ml to about 0.25 mg/ml. Thestable liquid formulation of the invention is preferably present in acontainer for single dosage administration such that that the total doseof the antagonist, preferably phentolamine mesylate, in the container isbetween about 0.02 mg to about 0.4 mg. More preferably, the containerhas between about 0.18 mg and about 0.43 mg of phentolamine mesylate,even more preferably between about 0.40 mg and about 0.43 mg ofphentolamine mesylate.

The preferred container for single dosages of the stable liquidformulations of this invention is selected from an ampule, a standarddental cartridge (e.g. a CARPULE) that fits into a standard dental localanesthetic syringe, or a pre-filled syringe. It is further preferredthat the container hold a volume of the stable liquid formulations ofthis invention of between about 1.6 to 1.8 mL. Standard dentalcartridges hold up to about 1.8 mL, but the amount of the stable liquidformulation in a dental cartridge according to this invention may varyslightly, depending upon the amount of headspace present after fillingthe cartridge.

It has now been discovered that stable liquid formulations comprisingalpha adrenergic receptor antagonists can be prepared which have a shelflife of at least 12 months at 25° C. The ability to store the antagonistin this fashion is preferable for use in a dental setting or othermedical situations, as the formulation is readily available and can beused directly.

In addition to the antagonist, the formulations of this inventioncomprise a solvent, which may be aqueous or a combination of organic andaqueous and a metal chelator. The formulations of this inventionoptionally comprise a buffer for maintaining pH, an antioxidant,surfactants, complexing agents and tonicity modifiers.

The solvent used in the formulations of this invention is typicallywater. In an alternate embodiment, a solvent such as, but not limitedto, glycerol, polypropylene glycol, mineral oil or polyethylene glycolis also present. The organic solvent, if present, is preferably at aconcentration of between about 5 to 40% (v/v) in water. The preferredsolvent is polypropylene glycol. More preferably, polypropylene glycolis present at a concentration of about 25% (v/v) in water.

The presence of a metal chelator is believed to be necessary to maintainstability of the formulation of this invention. The metal chelator ispreferably EDTA. When EDTA is employed it is preferably present in theformulation at a concentration of between about 0.5-2.5 mg/ml, morepreferably between about 0.5-1.0 mg/ml. Other metal chelators, such asdiammonium ethylenediamine triacetate, hydroxyethyl-ethylenediaminetriacetic acid, diethylenetriamine pentaacetic acid, nitriloacetic acid,and citric acid may be substituted for EDTA, preferably at an equivalentrange of molar concentrations.

The pH of an unbuffered aqueous formulation of this invention is betweenabout 4.0 to 6.0. The stability of the present formulations is notadversely affected at a pH as low as 2.0. Thus according to oneembodiment, the stable liquid formulation has a pH in a range of about2.0 to about 6.0, preferably in a range of about 2.0 to about 5.0, morepreferably in a range of about 3.0 to about 4.0, most preferably atabout 3.5. In an alternate embodiment, the pH of the formulation ispreferably between 3.5 and 4.5, more preferably between 3.8 and 4.2. Inorder to achieve a pH lower than that produced by simply solubilizingthe antagonist in a solvent in the presence of a metal chelator, an acidmust be added. In a preferred embodiment, the acid is either acetic acidor methane sulfonic acid. Acidified formulations are preferably bufferedwith a counter ion which is present in the range of about 1 mM to about100 mM, preferably about 10 mM to about 50 mM, most preferably about 10mM. The choice of counter ion is based upon the acid used to lower thepH of the formulation. Thus, when acetic acid is used to lower the pH,the preferred counter ion is sodium acetate. When methane sulfonic acidis used to lower the pH and phentolamine mesylate is the antagonist, themesylate serves as an appropriate counter ion and no additional counterion need be added. If the pH of the formulation is too low, NaOH may beadded to raise the pH back to the desired level.

Optional tonicity modifiers that may be present in the formulations ofthis invention include, but are not limited to, NaCl, d-mannitol anddextrose. When present, the tonicity modifier is preferably at aconcentration of between about 1 to 10% (w/v). Preferably, the tonicitymodifier is d-mannitol. More preferably, the d-mannitol is present at aconcentration of between about 4 to 5% (w/v).

Optional antioxidants present in the formulations of this inventioninclude, but are not limited to, ascorbic acid, sodium metabisulfite,butylated hydroxyanisole, and butylated hydroxytoluene. When present, anantioxidant is used at concentrations effective to carry out itsintended function. Such concentrations are well known to those of skillin the art of pharmaceutical formulations (see Remington'sPharmaceutical Sciences, A. Osol (ed.), 16th Edition, Mack PublishingCo., Easton, Pa. (1980)).

As an alternative to employing an antioxidant to enhance stability, thestable liquid formulation of this invention may be stored in a containersuch that a low level of oxygen is present in the headspace of thecontainer. Preferably, the headspace has less than 2% oxygen. Thereduced oxygen formulation can be prepared by purging the container withan inert gas, preferably nitrogen. In one embodiment of the invention,the reduced oxygen formulation is prepared by flushing an emptycontainer with an inert gas, filling the container with the stableliquid formulation while continually flushing with the inert gas, andsealing the container. See, e.g., U.S. Pat. No. 6,274,169.

Optional complexing agents, such as α-cyclodextrin or niacinamide mayalso be present in the formulations of this invention. When complexingagents are employed, they are used at concentrations effective to carryout their intended function. Such concentrations are well known to thoseof skill in the art of pharmaceutical formulations (see Remington'sPharmaceutical Sciences, A. Osol (ed.), 16th Edition, Mack PublishingCo., Easton, Pa. (1980)).

In a preferred embodiment, the formulation comprises a metal chelator(0.1 mg/ml to 10 mg/ml) and a tonicity modifier (1% to 10%). Mostpreferably, the formulation comprises disodium EDTA as a metal chelatorand d-mannitol as a tonicity modifier.

In a preferred embodiment, the stable liquid formulation is selectedfrom the formulations set forth below:

Formulation A Formulation B Formulation C Formulation D Formulation EFormulation F Ingredient (per mL) (per mL) (per mL) (per mL) (per mL)(per mL) Phentolamine 0.222 mg 0.222 mg 0.222 mg 0.222 mg 0.222 mg 0.222mg Mesylate, USP (Reliable Chemical) EDTA Na₂,  0.5 mg  1.0 mg  0.5 mg 1.0 mg  0.5 mg  1.0 mg USP D-Mannitol,   50 mg   50 mg   50 mg   50 mg  50 mg   50 mg USP Sodium  1.36 mg  1.36 mg — — — — Acetate, USP AceticAcid, q.s. to pH q.s. to pH — — — — USP 3.8 to 4.2 3.8 to 4.2Methanesulfonic — — — — q.s. to pH q.s. to pH Acid 3.5 to 4.5 3.5 to 4.5PPG WFI q.s. to 1.0 mL q.s. to 1.0 mL q.s. to 1.0 mL q.s. to 1.0 mL q.s.to 1.0 mL q.s. to 1.0 mL Formulation G Formulation H Formulation IFormulation J Formulation K Formulation L Ingredient (per mL) (per mL)(per mL) (per mL) (per mL) (per mL) Phentolamine 0.222 mg  0.1 mg  0.1mg  0.1 mg  0.1 mg 0.235 mg Mesylate, USP (Reliable Chemical) EDTA Na₂, 0.5 mg  0.5 mg  0.5 mg  0.5 mg  0.5 mg  0.5 mg USP D-Mannitol,   50 mg  50 mg   50 mg   44 mg —   50 mg USP Sodium  1.36 mg  1.36 mg  1.36 mg 6.80 mg  1.36 mg Acetate, USP Acetic Acid, q.s. to pH q.s. to pH q.s.to pH q.s. to pH q.s. to pH USP 3.5 4.0 3.5 3.5 3.8 to 4.2 PPG 259.5 mgWFI q.s. to 1.0 mL q.s. to 1.0 mL q.s. to 1.0 mL q.s. to 1.0 mL q.s. to1.0 mL q.s. to 1.0 mL

The term “about” includes the recited number +/−10%. Thus, “about 0.5”means 0.45 to 0.55.

In a further embodiment of the invention, the stable formulation can beprepared as a semi-solid formulation, for example as a gel or paste. Agel or paste can be a one-phase or two-phase system. Two-phase systemscan be made from bentonite. One phase systems can be made from syntheticmacromolecules (e.g., carbomer, methylcellulose, carboxymethylcellulose,polyvinyl alcohols) or from natural gums (e.g., tragacanth, sodiumalginate, gelatin). See Remington's Pharmaceutical Sciences, A. Osol(ed.), 16th Edition, Mack Publishing Co., Easton, Pa. (1980). Typicalgel formulations include polyethylene glycol with or without glycerin,polaxomer (15-50%) with or without glycerin, hydroxypropyl cellulose(around 4%) with a high molecular weight polyethylene glycol, propylenecarbonate with stearalkonium hectorate or stearalkonium chloride, orcolloidal silicone dioxide (2-10%). A typical paste formulation isAPHTHASOL, which comprises benzyl alcohol, gelatin, glycerylmonostearate, mineral oil, pectin, petrolatum, and sodiumcarboxymethylcellulose. All formulations may contain an antimicrobialagent (e.g., benzyl alcohol, EDTA, methyl and propyl paraben).Formulations for oral and dental use may also contain flavoring agents,such as bubble gum or cherry flavoring and sweeteners such as xylitol orsucrose. See, e.g., U.S. Pat. Nos. 6,447,755, 6,355,001, 6,331,291,6,312,669, 6,159,446, 5,908,612, and 5,670,138.

The invention also relates to a kit comprising a carrier means such as acarton or box having in close confinement therein two or more containermeans such as dental cartridges or CARPULEs, vials, tubes, jars and thelike. A first container means comprises an anesthetic agent andoptionally an alpha adrenergic receptor agonist and a second containermeans comprises a stable liquid formulation comprising a low dose of analpha adrenergic receptor antagonist. Alternatively, the alphaadrenergic receptor agonist may be present in a separate containermeans. A further container means may comprise hyaluronidase.Alternatively, the hyaluronidase is in the same container means as thealpha adrenergic receptor antagonist. In a preferred embodiment, theanesthetic agent, alpha adrenergic receptor agonist, alpha adrenergicreceptor antagonist and, optionally, the hyaluronidase are present in1.8 ml dental cartridges (CARPULES) that fit into a standard dentallocal anesthetic syringe. Such cartridges are available commerciallyfrom a variety of suppliers, e.g. Henry Schein, Port Washington, N.Y. Inthis embodiment, a cartridge containing the local anesthetic and alphaadrenergic receptor agonist is placed into the syringe, and the mixtureis injected. The cartridge may then be removed and a second cartridgeinserted which contains the alpha adrenergic receptor antagonist and,optionally, the hyaluronidase.

In a further embodiment, the kit comprises a first container meanscomprises an anesthetic agent and optionally an alpha adrenergicreceptor agonist and a second container means comprises a stable liquidformulation comprising phentolamine mesylate. The phentolamine mesylatemay be present at any concentration up to the solubility limit of thedrug.

The anesthetic agent, vasoconstrictor and, optionally, the hyaluronidasemay be present in solution, preferably, a sterile solution, optionallycontaining salts and buffers, or as part of a gel or paste for topicaladministration. See U.S. Pat. No. 4,938,970 and Remington'sPharmaceutical Sciences, A. Osol (ed.), 16th Edition, Mack PublishingCo., Easton, Pa. (1980).

Mammals which may be treated according to the present invention includeall mammals that may experience the beneficial effects of the presentinvention. Such mammals include without limitation humans and veterinarymammals such as cattle, pigs, sheep, horses, dogs, and cats. Whenapplied to children and veterinary animals, the prompt reversal ofanesthesia inhibits the child or animal from tearing open fresh sutures.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLES Example 1

Study Rationale and Purpose

Local anesthesia is widely used by dentists to effect anesthesia duringdental procedures. Local anesthetics often contain alpha adrenergicreceptor agonists to cause vasoconstriction thereby prolonginganesthesia. The vasoconstrictor is necessary because local anestheticswithout vasoconstrictor are too short-acting for most dental procedures.On the other hand, in many instances the prolonged local anestheticeffect lasts much longer than required for many dental procedures. Itwould be desirable to have a drug that could be used at will to rapidlyreverse local anesthesia after it is no longer needed. Lingering localanesthesia can be the cause of injuries due to biting of the tongue orlips. Lingering local anesthesia can also result in loss of productivitydue to missed work hours. Lastly, lingering local anesthesia is aninconvenience and it is perceived as an annoyance by many patients. Thepurpose of the present study was to determine whether phentolaminemesylate, an injectable alpha adrenergic receptor agonist, which is FDAapproved for the systemic treatment of hypertension in pheochromocytomapatients, rapidly reverses prolonged local anesthesia when injectedlocally at a very low concentration. The phentolamine mesylateconcentration chosen for the present study was so low that it would beexpected to lack systemic side-effects such as severe episodes ofhypotension that have been described with the high systemic drug doseswhich are approved by the FDA for the treatment of hypertension inpheochromocytoma patients.

Study Design

The present human subjects study was designed to determine whetherinjection of a physiological saline solution containing an extremely lowconcentration of phentolamine mesylate is able to accelerate thereversal of the effects of a previously injected local anesthetic agentcontaining an alpha adrenergic receptor agonist. An injection of thephysiological saline vehicle (without phentolamine mesylate) served asthe control. In order to compare the effects of phentolamine mesylate tothe vehicle in the same patient, bilateral local anesthesia injectionswere made into the mouth of the same patient. This was followed byinjection of the phentolamine mesylate containing local anestheticreversal agent (LARA) into one side of the oral cavity, and injection ofthe saline vehicle (control) solution into the opposite side of the oralcavity. The time to reversal of the local anesthetic effect on bothsides was then recorded to determine whether there is a differencebetween the two sides.

Drugs

The local anesthetic used was 2% polocaine (mepivacaine hydrochloride)with levonordefrin (1:20,000=0.05 mg/ml) (levonordefrin injection, USP)(Astra USA, Inc., Westborough, Mass. 01581). Levonordefrin is asympathomimetic amine with a pharmacological profile similar to that ofepinephrine, but with a lower potency. The local anesthetic reversalagent (LARA) was prepared as follows: A standard vial containing 5 mg oflyophilized phentolamine mesylate for injection, USP (BedfordLaboratories, Bedford, Ohio 44146) was reconstituted with 1 ml ofphysiological saline using a sterile, disposable 3 ml syringe and asterile disposable hypodermic needle. After dissolution of thelyophilized powder, 0.5 ml of the phentolamine mesylate solution waswithdrawn and injected into a 50 ml vial of physiological saline forinjection (USP) by means of a sterile disposable 3 ml syringe and asterile disposable hypodermic needle. The resulting LARA thus consistedof 0.05 mg/ml phentolamine mesylate in physiological saline.

Methods

Three healthy, male human subjects, age 34-50, volunteered to have localanesthetic injected in the mouth bilaterally under the lip in an easilyrepeatable location. The exact time of each injection was recorded. Theposition chosen was above (apical) the prominence of the root of theupper cuspid teeth. This is a common site selected to numb the cuspids,lateral incisors and upper lip. The volume of the local anestheticinjected was 1.7+0.1 ml on each side of the mouth. Twenty minutes afterthe local anesthetic was injected, each subject was re-injected with 1.6ml of LARA on one side and 1.6 ml of physiological saline on theopposite side. A different size needle was used for the anesthetic andLARA or saline. A longer needle (1¼″) was used for the local anestheticresulting in more solution being deposited around the infra-orbitalnerves. LARA or saline were injected with a shorter needle (½″)resulting in less LARA coming into contact with the anesthetic agentaround the infra-orbital nerves. After all subjects received anestheticagent followed by LARA or saline, the subjects were asked to test theintensity of numbness on both sides at the following sites in the mouthand face: teeth, nose, upper lip and gingiva. Numbness of the teeth wastested by biting or grinding. Lip numbness was tested with the touch ofthe finger or tongue, and nose numbness was tested with the touch of thefinger. Gingiva numbness was tested with the blunt end of a woodencotton swab.

Blinding

Two of the subjects (E and M) were blinded with respect to the side ofthe mouth where LARA or saline vehicle were injected, i.e. the subjectswere not told by the PI which side received LARA and which side receivedsaline vehicle. The third subject (H) was the PI of the study whoinjected himself. As a consequence, subject H was not blinded withrespect to the side at which LARA or saline were injected.

Results

In all three subjects there was a dramatic acceleration of localanesthesia reversal on the side that had been injected with LARAcompared to the side that had been injected with saline. No side-effectsof any kind were noted in any of the three subjects. In general, feelingto the teeth returned first. TABLE I shows the times at which numbnessdisappeared and sensations re-appeared in the three subjects at thevarious sites on both sides of the mouth and face. In the early stagesof recovery the subjects reported that it was somewhat difficult todetermine which side of the lip was recovering first. In the laterstages of recovery, however, the differences between the two sides ofthe lip were profound and dramatic. In the other parts of the mouth andface, lateral differences were reported to be pronounced even in thevery early stages of recovery. The difficulty to sense lateraldifferences in the lips between the two sides early in the recoveryprocess is thought to be due to the following fact: The labial branchesof the infra-orbital nerve decussate at the midline, resulting in acrossover of innervation (and resulting sensation) at the midline of theupper lip.

TABLE I Subject E - LARA on right hand side (RHS), Vehicle on LHSRecovery Time RHS Recovery Time LHS Site of anesthesia (Minutes)(Minutes) Teeth 80% Recovered 21 85 Teeth Fully Recovered 28 101 Nose 30143 Lip 41 83 Gingiva 46 141 Subject M - LARA on LHS, Vehicle on RHSRecovery Time LHS Recovery Time RHS Site of anesthesia (Minutes)(Minutes) Teeth 32 121 Nose 40 163 Gingiva 45 102 Lip 36 178 AllSensation 58 229 Subject H - LARA on RHS, Vehicle on LHS Recovery TimeRHS Recovery Time LHS Site of anesthesia (Minutes) (Minutes) Teeth 80%Recovered 19 201 Teeth 100% Recovered 27 218 Gingiva 42 137 Lip 37 226Nose 25 140 All Sensation 58 263

CONCLUSION

LARA had a profoundly faster effect on removing the numbness associatedwith local anesthesia than using physiological saline. The total amountof phentolamine-mesylate contained in the administered LARA solution was0.08 mg (1.6 ml of a 0.05 mg/ml solution). This total dose ofphentolamine-mesylate is approximately 62 times lower than the 5 mg doseapproved by the FDA for systemic treatment of hypertension inpheochromocytoma patients (1 ml of a 5 mg/ml solution) and which cancause severe episodes of hypotension in normal patients. At theextremely low efficacious doses found to be effective in the presentstudy, any systemic side effects, such as those that can occur with theFDA-approved high dose, are likely to be absent. Indeed, in the presentstudy, no side-effects of any kind were noted during or afteradministration of 0.05 mg/ml phentolamine mesylate.

Example 2

Study Rationale and Purpose

As discussed in Example 1, it is desirable to rapidly reverse localanesthesia after it is no longer needed, for example followingcompletion of a dental procedure. The results in Example 1 indicate thatadministration of a low dose of an alpha adrenergic receptor antagonistsuch as phentolamine mesylate can reverse the anesthesia due toadministration of a local anesthetic comprising an alpha adrenergicreceptor agonist. It would be convenient to have a stable liquidformulation of a low dose of the alpha adrenergic receptor antagonistwhich could be stored refrigerated or at room temperature and whichcould be used directly for administration. Unfortunately, prior artphentolamine mesylate formulations are unstable in water and saline. Thepurpose of the present study was to determine whether a stable liquidformulation comprising phentolamine mesylate could be developed whichwould provide a shelf life of at least 12 months at 2-8° C. or 25° C.

Study Design

The phentolamine mesylate formulation development was conducted in atiered approach to define the optimal composition for the drug. At eachtier, one of the critical stability-related formulation parameters wasevaluated and the optimal condition was selected. The parametersincluded pH, buffer species, buffer concentration, and additivesincluding antioxidants, metal chelators, surfactants, and complexingagents. At each tier, a temperature-accelerated stability study at 40°C. or 60° C. was performed in order to define the preferred conditionfor the parameter. Once optimized formulations were identified,real-time (2-8° C. or 25° C.) and accelerated (40° C., 60° C. and 80°C.) stability studies were performed.

Methods

Phentolamine mesylate (99%, Product Number: 36, 165-8) was acquired fromAldrich Chemical Company (Milwaukee, Wis.). This material was used forthe majority of the studies. Reference standard phentolamine mesylate(Lot I) was acquired from United States Pharmacopoeia (USP) for use inthe real-time and accelerated stability studies. Upon receipt, thephentolamine mesylate was stored at room temperature according to theCertificates of Analysis.

The HPLC analytical method for phentolamine mesylate is an isocraticelution method using 20 mM KH₂PO₄ in 45% methanol mobile phase at 1ml/min flow rate and a Synergi Max RP column. The drug is monitored at adetection wavelength of 232 nm.

1. Preparation of Phentolamine Mesylate Test Solution Samples forStability Evaluation

An appropriate amount of phentolamine mesylate was weighed (accurate to0.1 mg) and added to an aqueous vehicle containing the selected amountof buffer and/or additive to 0.1 mg/ml concentration. At thisconcentration, phentolamine mesylate is freely soluble at all studied pHvalues. Tightly sealed glass vials containing the test solutions wereplaced in an oven at 40° C. or 60° C. All samples were stored in thedark. Aliquots were removed at designated time points for HPLC analysisand pH measurement. All samples were analyzed without dilution.

2. Preparation of Phentolamine Mesylate Standard Solutions for HPLCAnalysis

Standard stock solutions were prepared by accurately weighing theappropriate amount (defined in the individual studies) of phentolaminemesylate drug substance (Aldrich Chemical Company) into a volumetricflask, and filling the flask to volume with the solutions specified ineach individual experiment procedure. All standard solutions were storedfrozen at −20° C. and thawed at room temperature before use.

3. pH Measurement

In conjunction with HPLC analysis, pH was measured. The measurement wasperformed using the same samples as used for the HPLC analysis. The pHmeter was calibrated using pH 4.00 and 7.00 buffer standards prior toeach measurement.

Results

The phentolamine mesylate formulation was developed by studyingstability according to the following parameters (tiers).

1. Wide-Range pH-Stability Profiling

This study was conducted to define the range of pH (2-3 units) in whichphentolamine mesylate would be most stable. Phentolamine mesylatesolutions at 0.1 mg/ml concentration were prepared at pH 2, 3, 4, 6, 7,and 8 in 50 mM sodium phosphate and at pH 4, 5, and 6 in 50 mM sodiumacetate. The phosphate buffers were prepared by adding phosphoric acid(3.40 ml) to de-ionized (DI) water, adjusting the pH with 2 N NaOH, andbringing the volume to 1 L in a volumetric flask. The acetate bufferswere prepared by adding acetic acid (2.95 ml) to water, adjusting the pHvalue with 2 N NaOH, and bringing the volume to 1 L in a volumetricflask.

Samples were analyzed by HPLC for phentolamine concentration and purityat an initial time point and after two and five days storage at 40° C.Measurements were performed for pH after samples had been analyzed byHPLC.

In all stability studies, glass serum vials with rubber closures andflip-top seals were used for storage of the samples to minimize fluidloss. The weights of samples were measured at the beginning of theexperiment, prior to sampling for analysis, and after resealing thevials to monitor for any weight loss due to heating. No significantweight loss was observed.

At each pH, the concentration was divided by the corresponding initialconcentration (day 0) to obtain the percent recovery. The percentrecovery was plotted against time for calculation of first-order ratekinetics. The slope of each pH line was calculated as the rate constantand plotted against pH to obtain a wide-range pH-stability profile.

A significant increase in rate of degradation was observed at pH above 7(FIG. 1). At pH values below 7, the profile seems less characteristic,suggesting the necessity for a narrow range pH profile to preciselydetermine a narrow pH range in which the drug is most stable. Thesamples prepared in acetate buffers appeared to be more stable thantheir counterparts in the phosphate buffers.

2. Narrow-Range pH-Stability Profiling

After defining the preferred pH range for phentolamine mesylate, thisstudy was conducted to define the most favorable pH range (within 1 pHunit).

Phentolamine mesylate solutions at 0.1 mg/ml concentration were preparedat pH 2, 2.5, 3, 3.5 or 4 in 50 mM sodium phosphate. The phosphatebuffers were prepared as previously described. The samples were analyzedby HPLC for phentolamine concentration and purity at an initial timepoint and after 2, 7 and 14 days of storage at 60° C. Measurement ofsample pH was done after HPLC analysis.

In order to precisely define the most favorable pH, a stronger stressingcondition was needed to generate a greater extent of degradation in thelower pH region. For this study, the incubation condition was shiftedfrom 40° C. to 60° C. TABLE II provides the concentration and purity ofphentolamine recovered at each time point. Since the extent ofdegradation after fourteen (14) days was still less than twenty percent(20%) in most samples, zero-order rate kinetics was used for thedegradation curves (concentration versus time). The calculated rateconstant was plotted against pH to provide the narrow-range pH-stabilityprofile.

It was demonstrated that phentolamine was most stable at pH 3.5 (FIG.2). A narrow-range of the most preferable pH values thus would bedefined as pH 3.0 to 4.0 (pH 3.5±0.5).

TABLE II A Narrow-range pH Stability Profile Study Sodium PhosphateBuffer pH Day 0 Day 2 Day 7 Day 14 Phentolamine Concentration (μg/ml) pH2.0 98 98 93 81 pH 2.5 99 96 78 79 pH 3.0 96 93 91 84 pH 3.4 97 95 96 91pH 4.0 97 95 94 89 Phentolamine Purity (% peak area) pH 2.0 100 99 96 92pH 2.5 99 97 92 86 pH 3.0 99 97 95 91 pH 3.4 99 98 97 94 pH 4.0 99 98 9796

3. Effect of Buffer Anion on Stability

Having defined the optimal pH range (pH 3 to 4) from the previous twostudies, the purpose of this study was to select the most suitablebuffer species for buffering phentolamine mesylate solution at theoptimal pH value. A comparison of several injectable anionic buffers wasmade based on the relative stability of phentolamine mesylate underaccelerated stability conditions in these buffers. The test buffersincluded 50 mM sodium phosphate, 50 mM sodium acetate, 50 mM sodiumlactate, 50 mM sodium citrate, and 50 mM sodium succinate buffers at pH3. The 0.1 mg/ml phentolamine mesylate solutions prepared in thesebuffers were incubated at 60° C. to accelerate the phentolamine mesylaterate of degradation.

The phosphate and acetate buffers were prepared as described above. Thecitrate buffer was prepared by adding 9.60 g of citric acid to water,adjusting the pH with 2 N NaOH to pH 3, and bringing the volume to 1 Lin a volumetric flask. The lactate buffer was prepared by adding 2.25 gof lactic acid to water, adjusting the pH with 2 N NaOH to pH 3, andbringing the volume to 1 L in a volumetric flask. The succinate bufferwas prepared by adding 2.95 g succinic acid to water, adjusting the pHwith 2 N NaOH to pH 3, and bringing the volume to 1 L in a volumetricflask.

The samples were analyzed by HPLC for phentolamine concentration andpurity at one initial time point and after 2, 7 and 14 days of storageat 60° C. Measurements of sample pH were done after HPLC analysis.

TABLE III provides concentration and purity of phentolamine recovered ateach time point. Sodium acetate appeared to be the superior buffer. Arank of buffer preference based on the amount of phentolamine recoveredat the end of the study may be generated as: sodium acetate>no buffer(water with pH adjusted to pH 3)>sodium phosphate>sodium lactate>sodiumsuccinate>sodium citrate. The strong effect of buffer species onphentolamine stability suggested that degradation of phentolamine iscatalyzed by anionic species with the rate of degradation correlatedwith the negative charge on the anion. A multi-valent anion such ascitrate should be avoided. Sodium acetate was selected for furtherstudy.

TABLE III Effect of Anionic Buffer Type on Phentolamine Stability (allsodium salt) at pH 3.0 Buffer Day 0 Day 2 Day 7 Day 14 PhentolamineConcentration (μg/ml) Phosphate 96 93 91 84 Acetate 96 95 94 89 Lactate97 94 91 83 Citrate 93 87 73 62 Succinate 96 88 74 64 PhentolaminePurity (% peak area) Phosphate 99 97 95 91 Acetate 100 98 97 96 Lactate99 98 94 89 Citrate 97 91 77 69 Succinate 99 92 78 74

4. Effect of Buffer Concentration on Stability

After the most suitable buffer species was established (sodium acetate),this study was conducted to determine the optimal buffer concentrationbased on phentolamine mesylate drug substance stability and stability ofthe pH (lack of drift) of the solution during storage.

Phentolamine mesylate solutions (0.1 mg/ml) were prepared in sodiumacetate buffer at concentrations of 0, 5, 10, and 50 mM at pH 3.5.Samples were incubated at 60° C. with scheduled testing performed at day0, 2, 7, and 14. The various buffer concentrations were prepared bydiluting the 50 mM buffer with water and re-examining the pH value todetermine if the pH 3.5 was constant. Measurements of pH were performedafter HPLC analysis of the samples.

The concentration and purity of phentolamine recovered from solutionsprepared at 0, 5, 10 and 50 mM sodium acetate are provided in TABLE IV.The rate of degradation of phentolamine at 5, 10 and 50 mM sodiumacetate appeared to be comparable and was an improvement over theun-buffered control (0 mM). An upward pH drift was observed in allsamples, ranging from 0.1 to 0.7 pH units. The pH value was most stablein the 50 mM buffer. While a pH drift beyond the most stable pH range(pH 3.0 to 4.0) would be undesirable, an excessively strong buffer wouldalso be undesirable because a strongly buffered acidic solution maycause tissue irritation or pain at the injection site. Thus, the 10 mMconcentration was considered the preferred buffer concentration for theformulation.

TABLE IV Effect of Buffer Concentration on Stability of PhentolamineMesylate in Sodium Acetate at pH 3.5 Buffer Phentolamine ConcentrationConcentration (μg/ml) (mM) Day 0 Day 2 Day 7 Day 14 50 103 101 99 96 10100 97 96 92  5 99 97 94 92  0 90 92 82 77 Buffer Phentolamine PurityConcentration (% peak area) (MM) Day 0 Day 2 Day 7 Day 14 50 98 98 98 9610 99 99 98 98  5 99 99 98 98  0 99 99 98 97 Buffer Concentration pH(mM) Day 0 Day 2 Day 7 Day 14 50 3.5 3.6 3.6 3.6 10 3.5 3.7 3.8 3.7  53.5 3.8 3.8 3.8  0 3.6 4.4 5.1 5.3

5. Effect of Additives on Stability

After defining the optimal pH, pH buffer species, and pH bufferconcentration for a phentolamine mesylate solution, various additiveswere tested for their potential for enhancing phentolamine stability,and for selection of the most compatible tonicity modifier.

The additives in this study are selected from five groups of injectableexcipients namely, tonicity modifiers (NaCl and d-mannitol), metalchelators (EDTA), antioxidants (sodium metabisulfite), complexing agentsα-cyclodextrin and niacinamide) and solvents (glycerol and polypropyleneglycol). A tonicity modifier is required for adjusting the phentolaminemesylate solution to isotonic. The metal chelator and antioxidant wereselected based on the hypothesis that oxidation of phentolamine mayoccur as suggested by its molecular structure. Complexing agents wereapplied in the hope that a possible formation of an inclusion complex(by α-cyclodextrin) or stacking complex (by niacinamide) withphentolamine would modify the rate of hydrolytic degradation of the drugsubstance. Solvents were added to reduce the dielectric constant of thevehicle and were intended to slow the rate of degradation due to solventpolarity.

Solid additives (disodium EDTA, sodium metabisulfite, niacinamide,α-cyclodextrin, mannitol, and sodium chloride) were added directly intothe solution of 0.1 mg/ml phentolamine mesylate in 50 mM acetate bufferpH 3.5. The pH value of the solutions was readjusted to pH 3.5, asrequired. The 25% glycerol (25 g in 100 ml total volume with water) and25% polypropylene glycol (25 g in 100 ml total volume with water)solutions had their buffer strength adjusted by addition of acetic acidfollowed by addition of 2 N NaOH for pH adjustment to pH 3.5 followed byaddition of the drug to 0.1 mg/ml.

All samples were placed in 10 ml glass serum vials, stoppered withrubber closures with an inert coating (Dalkyo Flurotec by WESTPharmaceutical), and sealed with flip-top caps after sampling. Vialswere placed at 60° C. after the weights were recorded.

Sampling occurred at the initial time point and on days 2, 7, and 14.Vials were weighed, sampled, sealed, re-weighed, and replaced under theselected storage condition until testing at the subsequent time point.Records indicate no significant weight loss during storage.

TABLE V lists the concentration and purity of phentolamine recovered ateach time point and the zero-order rate constant. Based on the rateconstant and purity value, sodium metabisulfite, NaCl, glycerol,polypropylene glycol and niacinamide were considered undesirable sincethey had negative impact on the recovery of the phentolamine compared tothe no-additive control. The impact of α-cyclodextrin appeared to benegligible based on purity value or perhaps slightly positive based onthe rate constant value. At 5% concentration, however, its use in theformulation was determined to be unjustified due to potential toxicity.d-Mannitol and disodium EDTA were found to improve stability based onthe rate constant or to exhibit no negative impact based on purityvalues. Both were selected for inclusion in the formulation, disodiumEDTA as stabilizer, d-mannitol as a tonicity modifier.

TABLE V Effect of Additives on Stability of Phentolamine Mesylate in 10mM Sodium Acetate, pH 3.5 Rate Phentolamine Concentration Constant(μg/ml) (μg/ml/ Additive Day 0 Day 2 Day 7 Day 14 day) EDTA, 106 103 10399 0.438 0.7 mg/ml Sodium metabisulfite, 97 101 84 74 1.903 0.5 mg/mlα-Cyclodextrin, 102 99 96 94 0.501 50 mg/ml NaCl, 94 98 80 74 1.663 50mg/ml Niacinamide, 90 90 78 92 −0.026 50 mg/ml d-Mannitol, 98 96 98 910.416 40 mg/ml Glycerol, 126 123 120 115 0.726 25% w/v Polypropyleneglycol, 118 121 113 106 1.019 25% w/v No additive 90 92 82 77 1.027Phentolamine Purity (% Peak Area) Additive Day 0 Day 2 Day 7 Day 14EDTA, 99 99 99 98 0.7 mg/ml Sodium metabisulfite, 97 99 95 92 0.5 mg/mlα-Cyclodextran, 98 99 98 98 50 mg/ml NaCl, 94 99 89 85 50 mg/mlNiacinamide, 99 100 93 90 50 mg/ml d-Mannitol, 96 97 96 97 40 mg/mlGlycerol, 98 98 93 94 25% w/v Polypropylene glycol, 93 97 93 90 25% w/vNo additive 99 98 98 97

6. Tonicity Testing and Adjustment

Samples were prepared with 2, 3, or 4% w/v d-mannitol with disodium EDTAat 0.5 mg/ml and 0.1 mg/ml phentolamine mesylate in either 10 or 50 mMacetate buffer. A vapor pressure osmometer (Westcore Model 5500) wasused to measure the osmolarity of the solutions as well as a control ofNormal Saline solution (0.9% sodium chloride, USP). The tonicity of thephentolamine solutions was represented by osmolarity. The measuredosmolarity was plotted against d-mannitol concentration. A linearrelationship was obtained between the osmolarity and d-mannitolconcentration. The concentration of d-mannitol required to achieve thesame osmolarity as the Normal Saline solution was calculated for the 10or 50 mM sodium acetate buffered solution.

TABLE VI lists the osmolarity values for the solutions including NormalSaline (0.9% sodium chloride), which at a measured osmolarity of 292mmole/kg is considered isotonic to body fluid. The concentration ofd-mannitol required to provide isotonicity was calculated to be 4.4% w/vand 5.0% w/v for the 50 mM and 10 mM acetate buffered solutions,respectively.

TABLE VI Osmolality of 0.1 mg/ml Phentolamine Mesylate with 0.5 mg/mldisodium EDTA in 10 or 50 mM sodium acetate buffer, pH 3.5 MeasuredOsmolality Calculated % Mannitol (mmole/Kg) for Isotonicity 10 mM 50 mM10 mM 50 mM sodium sodium sodium sodium % Mannitol acetate acetateacetate acetate 2 176 193 5.03 4.39 3 213 234 4 253 276 Normal 292Saline

7. Real Time and Accelerated Stability Testing

Upon completion of the studies as described above, a final phentolaminemesylate formulation (identified as formulation number 1) was defined asa 0.1 mg/ml phentolamine mesylate solution containing 0.5 mg/ml disodiumEDTA, 5.0% w/v d-mannitol in a 10 mM sodium acetate buffer, pH 3.5. Fourother formulations (numbers 2, 3, 4 and 5) were identified as listedbelow. Formulation numbers 2-5 were also included in this study forlong-term stability comparisons based on real-time stability data. Thereal-time stability is a stability profile generated at the preferredstorage temperature, i.e. 25° C. or 2-8° C. Stability data generated atelevated temperatures, i.e. 40, 60 and 80° C., could be used to generatean Arrehenius plot in order to predict long-term shelf-life at a lowertemperature, as needed. An acceptable shelf-life for a phentolaminemesylate solution formulation is defined as 90-95% recovery of labeledclaim of phentolamine mesylate after storage at 25° C. or 2-8° C. forone to two years.

Phentolamine Disodium Sodium Formulation Mesylate EDTA d-mannitolacetate pH 1 0.1 mg/ml 0.5 mg/ml 5% 10 mM 3.5 2 0.1 mg/ml 0.5 mg/ml 5%10 mM 4.0 3 0.1 mg/ml 0.5 mg/ml 4.4%   50 mM 3.5 4 0.1 mg/ml 0.5 mg/ml0% 50 mM 3.5 5 0.1 mg/ml   0 mg/ml 4.4%   50 mM 3.5

Formulations were prepared aseptically in a laminar flow hood. Thesolutions were sterilized by filtering through a 0.2-micron sterilefilter after the final pH adjustment. All formulations were sterilelyfilled into vials in a laminar flow hood with a repeat pipettor totransfer 1.5 ml of the filtered solution into 2 ml amber glass vials.Prior to filling, the vials were sterilized and depyrogenated by bakingat above 250° C. for approximately sixteen hours. The filled vials weresealed with autoclaved stoppers (13 mm finish gray butyl rubber stopper)and were then capped and weighed prior to placement in stability ovens.All vials were stored in the dark in an upright position. Triplicatevials for HPLC analysis at each time point were prepared and a singleHPLC injection for each vial was scheduled. Measurements of pH were tobe performed after HPLC analysis of a sample.

Formulation number one vials were placed at −20° C., 2-8° C., 25° C.,40° C., 60° C., and 75-82° C. Formulations 2, 3, 4 and 5 were placed at25° C. The intended time points for sampling and analysis were up to 48weeks for the 25° C. samples with truncated sampling schedules for the40° C., 60° C., and 75-82° C. samples.

During collection of the second week data, it was noted that the frozenstandard solution appeared to be less stable than the test samplesstored in the liquid state. A decision was made to switch the assaystandard from the frozen solution to solid phentolamine mesylate USPstandard.

USP reference standard was weighed into each of ten (10) volumetricflasks and stored dry for future use. At each time point, one of thesestandard aliquots was used to make a standard solution by adding 50 mmacetate buffer, pH 3.5 to the determined volume.

TABLE VII illustrates percentage recovery values of phentolamineconcentration at week 1, 2, 3, 4, 8, 12, 24, 36 and 48. For the time 0,week 1 and week 2 values, the phentolamine concentration values weredetermined by using a separately prepared frozen standard solution. Thisfrozen standard solution was later switched to solid phentolamine USPstandard for week 3 samples and later because of an apparent instabilityobserved in the frozen standard solution. Because of this standardswitch, an abrupt drop of 1-2% in phentolamine concentration wasobserved for the samples stored at 40° C. or below.

When a stable standard is used, a typical variation of two percent inconcentration is considered normal in HPLC analysis, thus an observedreduction in percentage recovery of less than two percent is notsignificant. The data in TABLE VII indicate that, at week 3, the totalobserved reduction in concentration was 4.0, 3.7, 3.8 and 4.7% for the−20° C., 2-8° C., 25° C. and 40° C. stored samples, respectively. Thedown shift of concentration between week 2 and week 3 due to standardchange could account for more than 50% of the total observed reductionin the concentration. Therefore, the loss of concentration in the first3 weeks is about 2% at most at 40° C. or below. A difference of 2% inconcentration could very well be within the HPLC method variation.

From week three to week eight, there appeared to be no loss ofconcentration of phentolamine in all samples stored at 40° C. or below.

The phentolamine concentration data for the 48-week time point shows4.6% and 3.2% degradation for samples stored at −20° C. and 2-8° C.,respectively. Samples stored at 25° C. showed 6.0% degradation, whilesamples stored at 40° C. showed 19.9% degradation. The samples stored at80° C. were completely degraded at the 12-week time point, and samplesstored at 60° C. were consumed after 24 weeks.

The concentration recovery values were well supported by the purityvalues (TABLE VIII). After 8 weeks, the loss of purity was less than twopercent for all samples stored at 40° C. or below. The purity data doesnot accurately reflect the sample purity since it appears that themajority of the degraded material elutes in the column's void volume andis not quantified.

The pH (TABLE IX) of all samples did not change significantly (≦0.2 pHunits) with the exception of the 60° C. and 80° C. stored samples. Anupper pH drift of about 0.5 pH unit was observed in the 80° C. storedsamples at week 8.

The stability study for formulations 2-5 was only conducted at 25° C.The 48-week concentration data for formulations 2, 3, and 4 show 5.3%,6.1%, and 4.8% degradation, respectively, similar to that observed forformulation 1. Formulation 5 showed 19.0% degradation. The maindifference between formulation 5 and the other four formulations is thatformulation 5 does not contain disodium EDTA.

TABLE VII Real-Time and Accelerated Stability Concentration Data forPhentolamine Formulations Week Week Week Week Time 0 Week 1 Week 2 Week3 Week 4 Week 8 12 24 36 48 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg.Avg. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc.Formulation Storage (μg/ml)* (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)(μg/ml) (μg/ml) (μg/ml) (μg/ml) 1 −20° C.   101.9 100.0 100.4 97.9 98.299.3 98.2 96.8 98.3 97.2 1 2-8° C.  100.1 101.0 98.2 98.6 100.0 98.397.4 99.2 98.6 1 25° C. 100.9 100.3 98.1 98.2 98.7 96.8 96.8 97.3 95.8 140° C. 100.2 99.8 97.2 97.4 97.2 94.8 88.8 88.2 81.6 1 60° C. 100.4 98.394.4 94.3 90.7 82.3 69.5 1 80° C. 91.2 76.9 58.7 54.4 4.2 0.6 2 25° C.100.2 99.6 98.7 96.7 97.2 98.1 95.7 94.8 96.6 94.9 3 25° C. 102.2 102.2101.9 98.2 99.2 99.2 97.9 96.9 95.8 96.0 4 25° C. 102.4 102.0 101.7 95.599.0 99.5 98.1 97.4 95.7 97.5 5 25° C. 101.9 99.3 96.2 94.0 94.9 93.289.8 89.0 85.0 82.5 *Average of three samples (n = 3)

TABLE VIII Real-Time and Accelerated Stability Purity Data forPhentolamine Formulations Week Week Week Week Time 0 Week 1 Week 2 Week3 Week 4 Week 8 12 24 36 48 Purity Purity Purity Purity Purity PurityPurity Purity Purity Purity (% Peak (% Peak (% Peak (% Peak (% Peak (%Peak (% Peak (% Peak (% Peak (% Peak Formulation Storage Area)* Area)Area) Area) Area) Area) Area) Area) Area) Area) 1 −20° C.   99.3 99.199.3 99.3 99.4 99.9 98.0 98.8 98.8 98.5 1 2-8° C.  99.4 99.2 99.2 99.499.2 98.3 98.6 98.7 97.8 1 25° C. 99.2 99.3 99.2 99.0 99.0 98.0 98.498.3 97.7 1 40° C. 99.1 99.0 98.8 98.8 98.2 96.8 93.4 94.1 90.6 1 60° C.99.5 97.8 97.5 97.4 95.6 93.2 86.8 1 80° C. 94.9 89.5 82.4 79.7 21.6 4.42 25° C. 99.2 99.1 99.1 99.1 99.1 99.0 97.9 98.1 98.1 97.4 3 25° C. 99.199.0 99.1 98.8 99.1 99.1 96.9 98.3 97.4 97.6 4 25° C. 98.7 98.5 98.799.0 98.7 98.4 96.7 97.7 97.2 96.6 5 25° C. 99.4 99.2 99.2 99.1 99.298.8 96.5 97.0 95.2 92.4 *Average of three samples (n = 3)

TABLE IX Real-Time and Accelerated Stability pH Data for PhentolamineFormulations Week Week Week Week Time 0 Week 1 Week 2 Week 3 Week 4 Week8 12 24 36 48 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg.Formulation Storage pH* pH pH pH pH pH pH pH pH pH 1 −20° C.   3.7 3.73.7 3.8 3.7 3.8 3.8 3.8 3.9 3.8 1 2-8° C.  3.7 3.7 3.8 3.7 3.8 3.7 3.83.8 3.7 1 25° C. 3.7 3.8 3.8 3.7 3.8 3.8 3.8 3.8 3.8 1 40° C. 3.7 3.83.8 3.8 3.9 3.8 3.9 3.9 3.9 1 60° C. 3.8 3.8 3.9 3.8 3.9 3.9 4.0 1 80°C. 3.8 3.9 4.0 4.0 4.2 4.3 2 25° C. 4.3 4.3 4.3 4.3 4.3 4.4 4.4 4.4 4.34.4 3 25° C. 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.8 3.7 3.7 4 25° C. 3.7 3.73.7 3.7 3.7 3.8 3.8 3.8 3.8 3.8 5 25° C. 3.6 3.6 3.6 3.6 3.6 3.6 3.7 3.73.6 3.7 *Average of three samples (n = 3)Conclusion

The 48-week data show phentolamine mesylate formulations 1-4 to berelatively stable at storage temperatures of 25° C. and lower. The datashow that formulation 1 stored at 2-8° C. should remain in specification(i.e., less than 10% loss in concentration and purity) for more than 180weeks, but has a predicted shelf life, based on the lower 95% confidencelimit, of 108 weeks. The data show that formulation 1 stored at 25° C.should remain in specification for 108 weeks, but has a predicted shelflife, based on the lower 95% confidence limit, of 69 weeks. The datashow that formulation 2 stored at 25° C. should remain in specificationfor 103 weeks, but has a predicted shelf life, based on the lower 95%confidence limit, of 63 weeks. The data show that formulation 3 storedat 25° C. should remain in specification for 88 weeks, but has apredicted shelf life, based on the lower 95% confidence limit, of 61weeks. The data show that formulation 4 stored at 25° C. should remainin specification for 115 weeks, but has a predicted shelf life, based onthe lower 95% confidence limit, of 60 weeks.

The rapid degradation of formulation 5 confirms that the presence ofdisodium EDTA is essential for a stable phentolamine formulation.

Example 3

The objective of this proof-of-principle trial was to evaluate thefeasibility of reversing soft-tissue anesthesia by pharmacologicallyblocking the effects of epinephrine. Twenty healthy adult volunteerswere given an inferior alveolar nerve block using one 1.8 ml cartridgeof 2% lidocaine with 1:100,000 epinephrine. Sixty minutes later, tensubjects received an injection of phentolamine mesylate for injection,USP (0.2 mg in 1.8 ml), reconstituted and diluted with 0.9% sodiumchloride for injection, USP, and 10 other subjects received an injectionof saline in the same site as the local anesthetic injection. Subjectsself-evaluated the return of normal sensation in their lip, chin,tongue, nose, and teeth by palpations at 5-minute intervals starting at5 minutes before the study drug injection and continuing forapproximately 4-5 hours. To avoid biased responding, subjects were toldthat they could not leave the clinic for 8 hours after the lastinjection. Responses for soft tissues were categorized as (1) numb (nofeeling), (2) feeling of pins and needles, or (3) normal sensation.Responses for teeth were (1) numb (no feeling) or (2) normal sensation.Safety was evaluated through reports of adverse events, measurement ofvital signs and two-lead electrocardiograms before, during, and afterthe injections of anesthetic and study drug, and through painassessments using visual analog scales.

Phentolamine mesylate significantly (p<0.01) reduced the duration ofanesthesia in each tissue. The average times from the injection of studydrug until the return to fully normal sensation are depicted in FIG. 3.Adverse effects were few and no differences were noted between theactive and placebo groups. There were no abnormalities in theelectrocardiogram.

Example 4

The objectives of this study were to evaluate the safety and efficacy ofa range of doses of phentolamine in reversing local anesthesia after amandibular nerve block. Four groups of ten healthy adult volunteersubjects were given an inferior alveolar nerve block using one 1.8 mlcartridge of lidocaine 2% with epinephrine 1:100,000. Sixty minuteslater, each group received an injection of phentolamine mesylate forinjection, USP (0.02 mg, 0.06 mg, or 0.4 mg in 1.8 ml), reconstitutedand diluted with 0.9% sodium chloride for injection, USP, or the salineplacebo in the same site as the local anesthetic injection. Subjectspalpated their lower lip, chin, tongue, and teeth every five minutes forthe next five hours after injections.

Phentolamine mesylate, at all doses tested, significantly (p<0.05)reduced the duration of anesthesia in each measured tissue. The averagetimes from the injection of study drug until the return to fully normalsensation are depicted in FIG. 4. Adverse events were few and nodifferences were noted between the active and placebo groups. There wereno abnormalities in the electrocardiogram.

Example 5

The objectives of this study were to evaluate the safety and efficacy ofa range of doses of phentolamine in reversing local anesthesia after amaxillary infiltration. Four groups of eight healthy adult volunteersubjects were given an infiltration of the lateral incisor using one 1.8ml cartridge of lidocaine 2% with epinephrine 1:100,000. Forty minuteslater, each group received an injection of phentolamine mesylate forinjection, USP (0.02 mg, 0.08 mg, or 0.4 mg in 1.8 ml), reconstitutedand diluted with 0.9% sodium chloride for injection, USP, or the salineplacebo in the same site as the local anesthetic injection. Subjectspalpated their upper lip, nose, and teeth every five minutes for thenext five hours after injections.

Phentolamine mesylate, at all doses tested, significantly (p<0.01)reduced the duration of anesthesia in each measured tissue. The averagetimes from the injection of study drug until the return to fully normalsensation are depicted in FIG. 5. Adverse events were few and nodifferences were noted between the active and placebo groups. There wereno abnormalities in the electrocardiogram.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A method for increasing blood flow in a mammal comprisingadministering a stable liquid formulation to said mammal, wherein saidstable liquid formulation consists essentially of: (a) a low dose of analpha adrenergic receptor antagonist, a buffer for maintaining pH, anaqueous solvent, a metal chelator, optionally a tonicity modifier,optionally an antioxidant, and optionally a complexing agent, whereinsaid formulation has a pH of from about 2.0 to about 6.0; or (b) analpha adrenergic receptor antagonist, a buffer for maintaining pH, anaqueous solvent, a metal chelator, optionally a tonicity modifier,optionally an antioxidant, and optionally a complexing agent, whereinsaid formulation has a pH of from about 2.0 to about 6.0, and whereinsaid stable liquid formulation is present in a dental cartridge or apre-filled sterile syringe.
 2. The method according to claim 1, whereinsaid administering a stable liquid formulation counteracts a prioradministration to said mammal of an alpha adrenergic receptor agonist.3. The method according to claim 1, wherein said administering a stableliquid formulation reverses anesthesia caused by prior administration ofan anesthetic.
 4. The method according to claim 3, wherein saidanesthetic was administered with an alpha adrenergic receptor agonisteither as a single dosage form or as a separate dosage form.
 5. Themethod according to claim 3, wherein said method is to reverseprolongation of local anesthesia administered as part of a dentalprocedure.
 6. A method according to claim 1, wherein said method is toenhance tissue graft survival, reduce dermal necrosis, decrease drysocket in a mammal undergoing tooth extraction, reduce post-operativepain, reduce muscle spasms, enhance the survival of an injured ordiseased tooth, treat a trigger point, reduce the prolongation of aregional anesthetic block or treat periodontal disease.
 7. The methodaccording to claim 6, wherein said method is to enhance tissue graftsurvival and wherein said method further comprises administeringhyaluronidase, wherein said hyaluronidase is administered following saidtissue graft.
 8. The method according to claim 6, wherein said tissuegraft is selected from a hair graft, a dental implant or a hair flap. 9.The method according to claim 6, wherein said method is to reduce theprolongation of a regional anesthetic block and comprises administrationinto the epidural space of said mammal.
 10. The method according toclaim 6, wherein said method is to treat a periodontal disease andcomprises administration as an irrigant for a periodontal pocket. 11.The method according to claim 1, wherein said alpha adrenergic receptorantagonist is selected from the group consisting of phentolamine,phentolamine hydrochloride, phentolamine mesylate, tolazoline,yohimbine, rauwolscine, doxazosine, labetalol, prazosine, tetrazosine,trimazosine, and pharmaceutically acceptable salts thereof.
 12. Themethod according to claim 11, wherein said alpha adrenergic receptorantagonist is phentolamine mesylate.
 13. The method according to claim1, wherein said alpha adrenergic receptor antagonist is present at aconcentration of between about 0.01 mg/ml and about 10 mg/ml.
 14. Themethod according to claim 13, wherein said alpha adrenergic receptorantagonist is present at a concentration of between about 0.1 mg/ml andabout 0.25 mg/ml.
 15. The method according to claim 14, wherein saidalpha adrenergic receptor antagonist is phentolamine mesylate.
 16. Themethod according to claim 1, wherein said aqueous solvent is water. 17.The method according to claim 1, wherein said metal chelator is selectedfrom the group consisting of diammonium ethylenediamine triacetate,hydroxyethyl-ethylenediamine triacetic acid, diethylenetriaminepentaacetic acid, nitriloacetic acid, citric acid, and EDTA.
 18. Themethod according to claim 17, wherein said metal chelator is EDTApresent at a concentration of between about 0.5 and about 1.0 mg/ml. 19.The method according to claim 18, wherein said alpha adrenergic receptorantagonist is phentolamine mesylate.
 20. The method according to claim1, wherein the pH of said formulation is between about 3.5 and about4.5.
 21. The method according to claim 1, wherein said pH is achievedwith a 10 to 50 mM acetate buffer, or with methanesulfonic acid.
 22. Themethod according to claim 21, wherein said alpha adrenergic receptorantagonist is phentolamine mesylate.
 23. The method according to claim1, wherein said stable liquid formulation contains a tonicity modifier.24. The method according to claim 23, wherein said tonicity modifier isselected from the group consisting of NaCl, d-mannitol, and dextrose.25. The method according to claim 24, wherein said tonicity modifier isd-mannitol present at a concentration of between about 4 and about 5%(w/v).
 26. The method of claim 25, wherein said alpha adrenergicreceptor antagonist is phentolamine mesylate.
 27. The method accordingto claim 1, wherein said stable liquid formulation contains anantioxidant in an amount sufficient to prevent oxidization of said alphaadrenergic receptor antagonist.
 28. The method according to claim 27,wherein said antioxidant is selected from the group consisting ofascorbic acid, sodium metabisulfite, butylated hydroxyanisole, andbutylated hydroxytoluene.
 29. The method of claim 28, wherein said alphaadrenergic receptor antagonist is phentolamine mesylate.
 30. The methodaccording to claim 1, wherein said stable liquid formulation contains acomplexing agent.
 31. The method according to claim 30, wherein saidcomplexing agent is selected from the group consisting ofα-cyclodextrin, and niacinamide.
 32. The method according to claim 31,wherein said alpha adrenergic receptor antagonist is phentolaminemesylate.
 33. The method according to claim 1, wherein said alphaadrenergic receptor antagonist is 0.235 mg/ml phentolamine mesylate,said metal chelator is 0.5 mg/ml EDTA, said tonicity modifier is 5%d-mannitol, and said buffer is 16.6 mM acetate buffer, wherein saidformulation has a pH of 3.8 to 4.2.